Extremely high purity oligonucleotides and methods of synthesizing them using dimer blocks

ABSTRACT

The present invention comprises an improved method of synthesizing oligonucleotides. The method comprises employing dinucleotides (or &#34;dimer blocks&#34;) as the basic synthetic unit building block. The method results in extremely high purity oligonucleotides in which the N-1 content is very low, generally less than 1-2% of the full length, N, oligonucleotide. We have found that synthesis using dinucleotide phosphorothioates results in oligonucleotides having very little phosphodiester content. Furthermore, we have found that the amount of dimer required in each coupling step can be less than about 6 and is preferably about 2 equivalents. Synthesis of oligonucleotides according to the dimer block approach described herein can also be conducted without the capping step that has heretofore been deemed necessary after each coupling.

This application is a continuation-in-part of U.S. application Ser. No.08/339,918, filed on Nov. 15, 1994, now abandoned, which is acontinuation of U.S. application Ser. No. 08/002,823, filed on Jan. 8,1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of chemical synthesis ofoligonucleotides. More particularly, the invention relates to thesynthesis of extremely high purity oligonucleotides.

2. Summary of the Related Art

Since the discovery by Zamecnik and Stephenson (Proc. Natl. Acad. Sci.75, 280 (1978)) that synthetic oligonucleotides can inhibit Rous sarcomavirus replication, there has been great interest in the use ofoligonucleotides and oligonucleotide analogs having modifiedinternucleotide linkages to control gene regulation and to treatpathological conditions. There have been many reports of successful useof antisense oligonucleotides to inhibit gene expression both in vitroand in vivo, either directly by binding to double stranded DNA, or,primarily, indirectly by inhibiting translation of mRNA.

Many reports of successful antisense inhibition of nucleic acidexpression in vitro have been reported. For example, Rapaport andZamecnik (U.S. Pat. No. 5,616,564) disclosed successful antisenseinhibition of malaria in parisitized erythrocytes. See also Barker etal. (Proc. Natl. Acad. Sci. USA 93, 514 (1996)).Oligodeoxyribonucleotide phosphorothioates have been found to inhibitimmunodeficiency virus (Agrawal et al., Proc. Natl. Acad. Sci. USA 85,7079 (1988); Agrawal et al., Proc. Natl. Acad. Sci. USA 86, 7790 (1989);Agrawal et al., in Advanced Drug Delivery Reviews 6, 251 (R. Juliano,Ed., Elsevier, Amsterdam, 1991); Agrawal et al. in Prospects forAntisense Nucleic Acid Therapy of Cancer and AIDS, 143 (E. Wickstrom,Ed., Wiley/Liss, New York, 1991); and Zamecnik and Agrawal in AnnualReview of AIDS Research, 301 (Koff et al., Eds., Dekker, New York,1991)), and influenza virus (Leiter et al., Proc. Natl. Acad. Sci. USA87, 3420-3434 (1990)) in tissue culture. In addition,oligodeoxyribonucleotide phosphorothioates have been the focus of a widevariety of basic research (e.g., Agrawal et al., Proc. Natl. Acad. Sci.USA 87, 1401 (1990) and Eckstein and Gish, Trends Biochem. Sci. 14, 97(1989)), enzyme inhibition studies (Mujumdar et al., Biochemistry 28,1340 (1989)), regulation of oncogene expression (Reed et al., CancerRes. 50, 6565 (1990)) and IL-1 expression (Manson et al., LymphokineRes. 9, 35 (1990)) in tissue culture. A number of review articles reportthe many published studies of successful antisense inhibition in vitro.E.g., Uhlmann and Peyman, Chem. Rev. 90, 543 (1990).

A number of published reports disclose the successful antisenseinhibition of nucleic acid expression in vivo. For example, Offenspergeret al. (EMBO J. 12, 1257 (1993)) demonstrated in vivo inhibition of duckhepatitis B virus. Nesterova and Cho-Chung (Nat. Med. 1, 528 (1995))demonstrated inhibition of tumor growth by a single subcutaneousinjection of antisense phosphorothioate oligonucleotide targeted to theRI, subunit of protein kinase A in nude mice. Several general reviews ofin vivo antisense inhibition have appeared that discuss these and otherstudies demonstrating successful in vivo antisense inhibition of nucleicacid expression as well as applications for therapeutic use. See, e.g.,Agrawal, TIBTECH 14, 376 (1996); Field and Goodchild, J. Exp. Opin.Invest. Drugs 4, 799 (1995).

These and other studies have proven sufficiently successful to justifyextension to humans. A number of human clinical trials are currentlyongoing, testing antisense oligonucleotides against a variety of diseasecausing targets, including HIV, CMV retinitis, ICAM, PKC, c-myb, andc-raf.

A necessary precursor to using antisense oligonucleotides to inhibitnucleic acid expression is the synthesis of the oligonucleotides.Various methods have been developed for the synthesis ofoligonucleotides for such purposes. Early synthetic approaches includedphosphodiester and phosphotriester chemistries. Khorana et al. (J.Molec. Biol. 72, 209 (1972)) discloses phosphodiester chemistry foroligonucleotide synthesis. Reese (Tetrahedron Lett. 34, 3143 (1978))discloses phosphotriester chemistry for synthesis of oligonucleotidesand polynucleotides. These early approaches have largely given way tothe more efficient phosphoramidite and H-phosphonate approaches tosynthesis. Beaucage and Caruthers (Tetrahedron Lett. 22, 1859 (1981))discloses the use of deoxynucleoside phosphoramidites in polynucleotidesynthesis. Agrawal and Zamecnik (U.S. Pat. No. 5,149,798) disclosesoptimized synthesis of oligonucleotides by the H-phosphonate approach.

Both of these modem approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild (Tetrahedron Lett. 28, 3539 (1987)) teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al. (Biochemistry 23, 3443 (1984)) disclosessynthesis of oligonucleotide phosphorothioates using phosphoramiditechemistry. Jager et al. (Biochemistry 27, 7237 (1988)) disclosessynthesis of oligonucleotide phosphoramidates using phosphoramiditechemistry. Agrawal et al. (Proc. Natl. Acad. Sci. USA 85, 7079 (1988))discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

A number of treatises and review articles have appeared that discuss thevarious synthetic approaches. E.g., Methods in Molecular Biology, Vol.20, Protocols for Oligonucleotides and Analogs, p. 63-80 (S. Agrawal,Ed., Humana Press 1993); Methods in Molecular Biology, Vol. 26:Protocols for Oligonucleotide Conjugates (Agrawal, Ed., Humana Press,Totowa, NJ 1994); Oligonucleotides and Analogues: A Practical Approachpp. 155-183 (Eckstein, Ed., IRL Press, Oxford 1991); Antisense Res. andApplns. pp. 375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, FL1993); Gene Regulation: Biology of Antisense RNA and DNA (Erickson andIzant, eds., Raven Press, New York, 1992).

Both phosphoramidite and H-phosphonate chemical syntheses are carriedout on a solid support that is stored in a reaction vessel. The requiredreaction steps for coupling each nucleotide are detritylation, coupling,capping, and oxidation. For small scale (up to 1 μmole) synthesis, thetotal time for the addition of one nucleotide is about 6 minutes. Anoligonucleotide, 30-mer in length, can be assembled in 180 minutes.Under these conditions, synthesized oligonucleotides are chemically pureand biologically active. However, when oligonucleotides are synthesizedon a larger scale (up to 1 mmole), the time for addition of eachnucleotide onto CPG is in the range of 30 to 60 minutes, requiringapproximately 12-25 hours for assembling a 25-mer oligonucleotide. Theincrease in time is due to the volume of the solid support being used insynthesis. This increase in cycle time exposes the already assembledoligonucleotide sequence to all reaction steps (including dichloroaceticacid detritylation step, coupling step, oxidation step and capping step)for a longer time. This increase in total assembly time affects theyield as well as chemical and biological properties of the compound. Thechemical and biological properties are mainly affected by depurination,base modifications, and the like.

To reduce the effects of these problems, it is possible to synthesizeoligonucleotides using dimeric or multimeric synthons, thereby reducingthe number of cycles, and thus the time required for synthesizingoligonucleotides. To this end, several investigators have worked towarddeveloping acceptable dimeric or multimeric synthon approaches. Khorana(Science 203, 614 (1979)) introduced the concept of multimeric synthons,using a phosphodiester approach. Crea and Itakura (Proc. Natl. Acad.Sci. USA 75, 5765 (1978)), Reese (Tetrahedron Lett. 34, 3143 (1978)),and Ohtsuka et al. (Nucleic Acids Res. 10, 6553 (1982)) all disclose useof dimeric or multimeric synthons in a phosphotriester approach. Kumarand Poonian (J. Org. Chem. 49, 4905 (1984)) and Wolter et al.(Nucleosides and Nucleotides 5, 65 (1986)) disclose synthesis ofoligonucleotide phosphodiesters using dimeric phosphoramidite synthons.Marugg et al. (Nucleic Acids Res. 12, 9095 (1984)) teaches use of adinucleotide thiophosphotriester to produce oligonucleotides containingone phosphorothioate linkage. Connolly et al. (Biochemistry 23, 3443(1984)) and Cosstick and Eckstein (Biochemistry 24, 3630 (1985))disclose addition of one dinucleotide phosphorothioate to a growingoligonucleotide chain using a phosphoramidite approach. Brill andCaruthers (Tetrahedron Lett. 28, 3205 (1987)) discloses synthesis ofthymidine dinucleotide methylphosphonothioates. Roelen et al. (NucleicAcids Res. 16, 7633 (1988)) discloses a solution phase approach, using areagent obtained in situ by treating methylphosphonothioic dichloridewith 1-hydroxy-6-trifluoromethyl benzotriazole to introduce amethylphosphonothioate internucleotide linkage into a dinucleotide in60-70% yield, and produces a hexamer containing the linkage by twoconsecutive condensations of dimers. Roelen et al. (Tetrahedron Lett.33, 2357 (1992)), discloses reagents for alkylphosphonate andalkylphosphonothioate chemistry. It discloses the solution phasesynthesis of TG methyl, n-butyl, and n-octyl phosphonate andphosphonothioate dimers.

Leba dev et al. (Tetrahedron. Lett. 31, 855 (1990)) discloses a solutionphase approach to produce dinucleotides containing a stereospecificmethylphosphonothioate internucleotide linkage in 50-60% yield. Kattiand Agarwal (Tetrahedron Lett. 27, 5327 (1986)) discloses 3'1-methoxycarbonate methylphosphonate dimers.

The use of such synthons in the synthesis of oligonucleotides has alsobeen disclosed. Kumar and Poonian, supra, demonstrated the use of 3'phosphoramidite methyl phosphotriester dimers in the solid phase manualsynthesis of a 29-mer with an overall yield of 93.4%. Wolter, supra,demonstrated the automated, solid phase synthesis of a 101-mer usingβ-cyanoethyl-protected phosphoramidite dimers. Miura et al. (Chem.Pharm. Bull. 35, 833 (1987)) discloses automated solid-phase synthesisof pentadecathymidilate with phosphoramidite dimers. And Bannwarth(Helv. Chim. Acta 68, 1907 (1985) disclosed the use of phosphoramiditedinucleotides in the synthesis of oligonucleotides of modest length(N=8-11).

Krotz et al. recently reported the synthesis of phosphorothioate dimershaving low phosphodiester dimer content. They used these dimers tosynthesize phosphorothioate oligo(T) and oligo(TC) nucleotides, whereinthey observed that the N/N-1 ratio was on the order of 99:1 as measuredby capillary gel electrophoresis (CGE). The phosphodiester content ofthe oligomers was on the order of 1% as determined by ³¹ P NMR foroligomers synthesized with phosphorothioate dimers wherein thephosphorothioate linkage is protected by a β-cyanoethyl group on thenon-bridging oxygen. A similar reduction of the phosphodiester contentwas not observed for dimers wherein the phosphorothioate linkage wasprotected by a β-cyanoethyl group on the (non-linking) sulfur.

Once synthesized, the desired oligonucleotide (being "N" nucleotides inlength) must be isolated from failure sequences (i.e., sequences withfewer than "N" nucleotides, such as N-1, N-2, etc.) and otherimpurities. While automated synthesizers have proven an invaluable toolfor obtaining oligonucleotides, 1-3% of the reactions fail during eachcycle in which a nucleotide monomer is to be added. Consequently, theresulting products are generally a heterogenous mixture ofoligonucleotides of varying length. For example, in a typical 20mersynthesis, the 20mer product represents only 50-80% of the recoveredoligonucleotide product.

Furthermore, preparation of oligodeoxynucleotides on a solid phasesupport requires that the oligodeoxynucleotide be cleaved from thesupport. Cleavage of the oligonucleotide from the support is typicallyaccomplished by treating the solid phase with concentrated ammoniumhydroxide. The ammonium hydroxide is conventionally removed underreduced pressure using, for example, a rotary evaporator. This methodfor removing the ammonium hydroxide, however, is not ideal for use inlarge scale isolation of oligodeoxynucleotides.

For most purposes (e.g., therapeutic or diagnostic) the purity of thecompounds is extremely important. Consequently, there has been aninterest in developing chromatographic techniques for purifyingoligonucleotides. Because of their therapeutic potential, much of thefocus has been on purifying oligonucleotide phosphorothioates.

Conventional methods for purifying oligodeoxynucleotides employreverse-phase liquid chromatography. Manufacturing facilities using suchmethods require explosion-proof equipment because acetonitrile istypically used in the elution buffer.

Methods of oligodeoxynucleotide phosphorothioate purification have beenpublished. Metelev and Agrawal (Anal. Biochem. 200, 342 (1992)) reportedthe ion-exchange HPLC analysis of oligodeoxyribonucleotidephosphorothioates on a weak anion-exchange column (Partisphere WAX) inwhich the weak anion exchanger utilizes a dimethylaminopropyl functionalgroup bonded to Partisphere silica. This medium, with an ion-exchangecapacity of 0.18 meq/g, exhibits an interaction with anions weaker thanthose observed with strong anion-exchange media. The authors of thisstudy found that separation was length dependent for oligonucleotidephosphorothioates up to 25 nucleotides in length. Furthermore, N-1 peakswere separated from the parent peak. They also found that 30-mer and35-mer oligonucleotide phosphorothioates were separable with the samegradient, although better separation could be obtained with a shallowergradient.

Metelev et al. (Ann. N.Y Acad. Sci. 660, 321-323 (1992)) reported theanalysis of oligoribonucleotides and chimericoligoribo-oligodeoxyribonucleotides using ion-exchange HPLC. They foundthat the retention time of the oligonucleotides studied depended on thenumber of ribonucleotide moieties in the oligonucleotide. In addition,the retention time of oligoribonucleotides was found to be lengthdependent. The authors noted that oligoribonucleotides of length up to25 nucleotides could be purified and analyzed.

Bigelow et al. (J. Chromatography 533, 131 (1990)) reported the use ofion-pair HPLC to analyze oligonucleotide phosphorothioates. Stec. et al.(J. Chromatography 326, 263 (1985)) and Agrawal and Zamecnik (NucleicAcids Res. 19, 5419 (1990)), reported HPLC analysis ofoligodeoxyribonucleotides containing one or two phosphorothioateinternucleotide linkages using a reversed-phase column.

Tang et al. (WO 95/27718) disclosed a purification techniques suitablefor large scale separation of oligonucleotide phosphorothioates. Themethod uses DMAE Fractogel EMD column with an organic solvent-free, lowsalt, elution buffer. The method does not require elevated temperatures,making it more amenable for large scale chromatography.

Puma et al. (WO 96/01268) disclosed a purification method not requiringthe removal of ammonium hydroxide or the use of conventional C-18 silicagel reverse-phase liquid chromatography. The disclosed methods usehydrophobic interaction chromatography and DEAE-5PW anion ion-exchangechromatography.

As antisense oligonucleotides proceed through human clinical trials,there is an ever-increasing demand for extremely pure oligonucleotidesin large quantities. Regulatory agencies around the world are addressingthe requisite standards for antisense oligonucleotides as drugcompounds. E.g., Kambhampati et al, Antisense Res. Dev. 3, 405 (1993).Consequently, there remains a need for new methods of producing largequantities of highly pure oligonucleotides.

SUMMARY OF THE INVENTION

The invention provides new methods for producing dimeric nucleotidesynthons, hereafter called "dimer blocks," having modifiedinternucleotide linkages, e.g,. alkylphosphonate, phosphoramidate,phosphorothioate, or alkylphosphonothioate. Phosphorothioates are thepreferred modified internucleotide linkage. According to this aspect ofthe invention, synthesis of dimer blocks proceeds in a single potsolution phase reaction, regardless of the type of internucleotidelinkage in the dimer block. For example, to synthesize dimer blockalkylphosphonates, condensation of a nucleoside 3'-alkylphosphonamiditewith a 3'-protected nucleoside is carried out. For synthesis of dimerblock phosphoramidates, alkylamine is added after H-phosphonatecondensation of nucleotides. For synthesis of dimer blockphosphorothioates, sulfurization using an appropriate sulfur reagentfollows solution phase coupling of the protected monomeric nucleotidesto yield a dimer. For preparing dimer block alkylphosphonothioates, analkylphosphonamidite is used in the same one pot reaction as describedfor dimer block phosphorothioates. This simple chemistry allows for thesynthesis of all possible dimer block methylphosphonothioates andpromotes preparation of dimer blocks having 3'-condensing groups.

Thus, in a second aspect the invention provides novel dimer blockscomprising the nucleotides GG, GA, GT, GC, AG, AA, AT, AC, TG, TA TC,TT, CG, CA, CT or CC linked together by alkylphosphonate,phosphoramidate, phosphorothioate or alkylphosphonothioate linkages, andhaving various combinations of protective groups and condensing groups.These dimer blocks also give rise to a method of using such dimer blocksto assemble oligonucleotides containing alkylphosphonate,phosphoramidate, phosphorothioate, or alkylphosphonothioate linkages.Moreover, the dimer blocks allow assembly of oligonucleotides havingexclusively alkylphosphonate, phosphoramidate, phosphorothioate oralkylphosphonothioate internucleotide linkages, or mixtures thereof.

Thus, in a third aspect, the invention provides methods of using dimerblocks to assemble oligonucleotides having alkylphosphonate,phosphorothioate, phosphoramidate or alkylphosphonothioate linkages orhaving combinations of two or more of these. It is an object of theinvention to provide efficient methods that reduce total assembly timeof oligonucleotides. It is a further object of the invention to provideefficient methods that reduce total solvent consumption required foroligonucleotide assembly. It is also an object of the invention toprovide efficient methods that ease purification of oligonucleotides byincreasing the yield of full length oligonucleotides. It is anadditional object of the invention to provide efficient methods thatreduce side reactions by reducing the exposure of partially assembledoligonucleotides to chemicals. It is also an object of this aspect ofthe invention to provide highly pure dimers that facilitate synthesisand purification of extremely highly pure oligonucleotides. Finally, itis an object of the invention to reduce overall cost of oligonucleotideassembly by allowing the use of inexpensive solution phase chemistry toachieve half of the total synthesis.

The present invention also provides oligonucleotides and methods forsynthesizing them with a heretofore unobtainable purity. In one aspect,the invention provides a population of oligonucleotides having a purityof greater than 98%. In one embodiment of this aspect of the invention,oligonucleotides are provided that have an N-1 content of less than 2%,preferably less than 1%, and more preferably less than 0.5% of thecontent of the desired oligonucleotide (the N oligonucleotide).

In one embodiment, the present invention comprises a population ofoligonucleotides having all phosphorothioate internucleoside linkages,wherein the amount of phosphodiester impurity at each of thephosphorothioate linkages is less than 1%, preferably less than 0. 1%,and most preferably undetectable, by ³¹ P NMR.

The extremely high purity of oligonucleotides according to the inventionsets a new industry standard.

In another aspect, the present invention comprises a method for largescale (≧ca. 100 μmol) synthesis of oligonucleotides of extremely highpurity. The method comprises synthesizing oligonucleotides usingnucleotide dimers (herein called "dimer blocks") in place ofmononucleotides as the basic synthetic unit. In the method of thepresent invention, oligonucleotide populations with extremely highpurity are obtained.

In another embodiment of this aspect of the invention, synthesis isconducted using standard techniques except that dimer blocks are usedand the normal capping step is eliminated. We have found that excellentresults (i.e., high yields of high purity oligonucleotide) can beobtained without the usual capping step.

In another embodiment of this aspect of the invention, synthesis byeither of the two previous embodiments is conducted using about six orless equivalents of dimer per coupling. Preferably, four equivalents andmost preferably two equivalents of dimer are used per coupling step.

The inventive method provides extremely high purity oligonucleotideswith both a savings in cost of production as well as time. Theoligonucleotides produced by the inventive method are ideally suited forin vivo therapeutic methods of treatment.

The foregoing merely summarizes certain aspects of the present inventionand is not intended, nor should it be construed as limiting theinvention in any way. All patents and other publications recited hereinare hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to reagents and methods for assemblingoligonucleotides. More particularly, the invention relates to theassembly of oligonucleotides having modified internucleotide linkages.

Synthesis of Dimer Blocks

In a first aspect, the invention provides new processes for making dimerblocks. Dimer blocks are dimeric nucleotides having modifiedinternucleotide linkages and blocking groups at the 5'-hydroxyl.Preferred blocking groups include tert-butyldimethylsilyl,dimethoxytrityl, levulinyl, monomethoxytrityl and trityl groups. The 3'position of the dimer block may have a blocking group, a free hydroxyl,or a β-cyanoethylphosphoramidite group. Preferred modifiedinternucleotide linkages include phosphorothioate, alkylphosphonate andalkylphosphonothioate linkages. The modified linkage of the dimer blockhas an alkoxy or alkyl group.

In a very general sense, the method of synthesizing dimer blocksaccording to the invention can be considered to be a method ofsynthesizing a dimer block having an alkylphosphonate, phosphoramidate,phosphorothioate or alkylphosphonothioate internucleotide linkage, themethod comprising the steps of:

(a) condensing together a first nucleoside derivative having aprotective group at a 5' end and a condensing group at a 3' end with asecond nucleoside derivative having a protective group at a 3' end and ahydroxyl group at a 5' end to form a dinucleotide derivative having areduced internucleotide linkage, and

(b) oxidizing the internucleotide linkage with an appropriate oxidizingagent to yield a dimer block alkylphosphonate, phosphoramidate,phosphorothioate or alkylphosphonothioate.

The precise dimer block obtained, of course will depend upon the natureof the first nucleoside derivative and the oxidizing agent, as shown inTable 1:

                  TABLE 1                                                         ______________________________________                                                   First Nucleoside                                                   Dimer Block Type                                                                         Derivative       Oxidizing Agent                                   ______________________________________                                        alkylphosphonate                                                                         nucleoside-3'-alkyl N,N di-                                                                    iodine                                                       isopropyl phosphonamidite                                          phosphoramidate                                                                          nucleoside-3'-H phosphonate                                                                    alkyl- or arylamine                               phosphorothioate                                                                         nucleoside-3'-O-alkyl N,N,                                                                     sulfurizing reagent                                          diisopropyl phosphoramidite                                        alkylphosphono-                                                                          nucleoside-3'-alkyl N,N                                                                        sulfurizing reagent                               thioates   diisopropyl phosphonamidite                                        ______________________________________                                    

In a first embodiment of this aspect of the invention, the methodproduces a dimer block having 5' and 3' blocking groups and aphosphorothioate internucleotide linkage. In this embodiment, the methodcomprises the steps of (a) joining together, by phosphoramiditechemistry, a nucleoside having a 5' blocking group and a nucleosidehaving a 3' blocking group, and (b) adding an appropriate sulfurizingagent, such as the Beaucage reagent. The Beaucage reagent(3H-1,2-benzodithiol-3-one-1,1-dioxide) is taught in U.S. Pat. No.5,003,097.

Examples of the preferred method of synthesizing phosphorothioate dimersare given below. We have found that synthesis of phosphorothioate dimersby this method results in a population of phosphorothioate dimers havingvery low phosphodiester dimer impurity. The use of thesephosphorothioate dimer blocks in the synthesis of oligonucleotides inturn results in phosphorothioate oligonucleotides with lessphosphodiester impurity.

In a second embodiment of this aspect of the invention, the methodproduces a dimer block having a 5' blocking group, a 3' free hydroxylgroup, and a phosphorothioate internucleotide linkage. In thisembodiment, the method comprises steps (a) and (b) of the firstembodiment above, and further comprises the step of (c) deprotecting the3'-hydroxyl group. This is achieved by the use of conditions selectivefor removal of the 3' protective group only. For example, if the 5'protective group is dimethoxytrityl, monomethoxytrityl or trityl, andthe 3' group is tert-butyldimethylsilyl, then selective removal of the3' group is obtained by treatment with tetrabutylammonium fluoride.Alternatively, if the 5' group is dimethoxytrityl, monomethoxytrityl ortrityl and the 3' group is levulinyl, selective removal of the 3' groupis obtained by treatment with hydrazine monohydrate in pyridine/aceticacid.

In a fourth embodiment of this aspect of the invention, the methodproduces a dimer block having a 5' blocking group, a 3' β-cyanoethylphosphoramidite group and a phosphorothioate internucleotide linkage. Inthis embodiment the method comprises steps (a), (b) and (c) of the firsttwo embodiments described above, and further comprises the step of (d)converting the free 3' hydroxyl group to a 13-cyanoethyl phosphoramiditegroup.

Those skilled in the art will recognize that as an alternative to thethird and fourth embodiments, dimer blocks having phosphotriester 3'groups can be prepared according to well known procedures.

In additional embodiments of this aspect of the invention the methodproduces dimer blocks having a 5' blocking group, analkylphosphonothioate, alkylphosphonate or phosphoramidateinternucleotide linkage, and a 3' group that may be a blocking group, afree hydroxyl, a β-cyanoethyl phosphoramidite group, or aphosphotriester group. In these embodiments, the method is carried outexactly as described for the four embodiments above to produce dimerblock alkyl-phosphonothioates, except that the starting material is anucleoside alkylphosphonamidite. Analogous dimer block alkylphosphonatesare prepared in identical fashion to the dimer blockalkylphosphonothioates, except that an iodine solution is used in placeof the sulfurizing agent. Analogous dimer block phosphoramidates areprepared by H-phosphonate condensation followed by oxidation of thelinkage with an alkyl- or arylamine in carbon tetrachloride.

This first aspect of the invention offers a method of producing dimerblock products that are useful as intermediates for assemblingoligonucleotides having modified internucleotide linkages. The abilityto produce these dimer blocks in a one pot reactions greatly simplifiestheir production.

Dimer Blocks

In a second aspect, the invention provides novel dimer block productshaving a 5' blocking group, a modified internucleotide linkage and a 3'group that may be a blocking group, a free hydroxyl, an H-phosphonategroup, or in some cases a β-cyanoethyl phosphoramidite. The method forproducing these dimer blocks is independent of the sequence of thenucleotides in the dimer block, thus allowing production of all possibledimer sequences containing alkylphosphonate, phosphoramidate,phosphorothioate or alkylphosphonothioate linkages, i.e., GG, GA, GT,GC, AG, AA, AT, AC, TG, TA, TT, TC, CG, CA, CT, and CC. Such dimers areillustrated below: ##STR1## Where B₁ and B₂ are the same or differentnucleotide base (e.g., G, A, T, C, or modifications thereof, and R₁ is aprotective group such as dimethoxytrityl, monomethoxytrityl or trityl, Lis an alkylphosphonate, phosphoramidate, thiophosphotriester, oralkylphosphonothioate, and R₂ is an H, a β-cyanoethylphosphoramidite, ora protective group such as levulinyl or t-butyldimethylsilyl. Althoughthe foregoing illustrates DNA/DNA dimers, RNA/RNA, RNA/DNA, and DNA/RNAdimers are also encompassed within the scope of the invention disclosedherein.

Synthesis of Ultra-pure Oligonucleotides Using Dimer Blocks

In a third aspect, the invention provides a method of using dimer blocksto assemble oligonucleotides having modified internucleotide linkages.In this aspect, dimer blocks having modified internuclcotide linkages(e.g., phosphorothioate, alkylphosphonate, phosphoramidate, oralkylphosphonothioate), are used to assemble oligonucleotides havingsuch modified internucleotide linkages (dimer blocks having 5' blockinggroups and 3' β-cyanoethyl phosphoramidite are used). Synthesis is thenconducted according to the phosphoramidite approach by condensing thedimer block with the nascent oligonucleotide. (As used herein, the term"nascent oligonucleotide" means the less-than-full-length, solidsupport-bound synthetic nucleic acid that upon elongation results in thedesired synthetic oligonucleotide.) Support of oligonucleotide synthesiswith dimer blocks can be, for example, soluble polymers as well asinsoluble CPG and polymer beads.

In order to ensure obtaining oligonucleotides of extremely high purityas disclosed herein, it will be appreciated that measures should betaken at each step in the synthetic/purification process to maximize thepurity. According to the present invention, this begins with usingdimers as the elemental oligonucleotide building block. In the syntheticmethods disclosed herein, dimer phosphoramidites are used. Preferablythe dimer phosphoramidite is at least 90% pure (as determined by HPLC).More preferably it is at least 96% pure. Most preferably it is at least98% pure.

In the phosphoramidite approach, β-cyanoethyltetraisopropylphosphorodiamidite and tetrazole are used to activate a5'-DMT, β-cyanoethyl protected dimer to yield the phosphoramidite dimer,such as depicted in the following in which a phosphorothioate dimer isillustrated: ##STR2## Alternatively, 2-cyanoethyldiisopropylchlorophosphoramidite can be used instead of the diamiditedisplayed in the scheme above.

Three principal types of impurities generally arise in this reaction.The first results from hydrolysis of the dimer amidite to yield anH-phosphonate dimer. The second impurity is the starting materialitself, the 3'-hydroxy dimer. Both of these impurities are "inert" inthe sense that they will not react with the nascent oligonucleotide andcontribute to the N-x or N+x content of the final oligonucleotideproduct (where N is the number of nucleotides in the final, full-lengtholigonucleotide and x is an integer≧1).

The third principal type of impurity is the tetrazole-activatedphosphorodiamidite. This impurity is not inert and measures should betaken to minimize its presence. Preferably, the tetrazole and dimer arefirst combined and then added together to the diamidite under conditionsin which the tetrazole is not in great excess of the diamidite. Thisresults in a low probability that two molecules of tetrazole will reactwith the diamidite. Preferably, the tetrazole and diamidite are used in˜1:1 ratio and each in slight excess of the dimer. A suitable amount ofeach is 1.3 cq diamidite and 1.2 eq tetrazole per eq of dimer. After thereaction is complete, the product is preferably washed several times asdescribed in the synthesis of dimer 24 in Example 4, below. Followingthe foregoing protocol minimizes the amount of non-inert impurity.

An advantage of the present method is that oligonucleotide synthesis canbe conducted on commercially available synthesizers using standardcycles, substituting dimers for the usual monomers. This isdemonstrated, for example, in Example 6-9, below.

Following synthesis, the oligonucleotide product is cleaved from thesolid support and subject to purification by Ion-Exchange Chromatography(IEX). Optionally, the oligonucleotide can first be purified by ReversePhase Chromatography (RPC) before IEX. As described by Puma et al. (WO96/01268), Hydrophobic Interaction Chromatography (HIC) can be useful asthe initial chromatographic step. For the purposes of the presentinvention, however, RPC is preferred. Advantageously, standard protocolsof purifying oligonucleotides by these techniques can be employed.Although High Pressure Liquid Chromatography (HPLC) can also be used,the present method offers the additional advantage of using MediumPressure Liquid Chromatography (MPLC), which is preferable because it ischeaper and more easily adaptable to large scale synthesis.

To obtain the high purity levels as disclosed herein, careful screeningand pooling of chromatography fractions is preferable. Screening offractions and/or trial pools of fractions is preferably conducted byanalytical Capillary Electrophoresis (CE) to determine the degree ofpurity and yield of each fraction. Then, fractions having an acceptabledegree of purity and yield are pooled in a pre-determined manner toyield a final population of oligonucleotides having a sufficiently highdegree of purity and yield. Depending on the application, one may bewilling to sacrifice some yield to obtain a higher degree of purity, andvice versa. Such pooling methods are well known to those skilled in theart.

Following this general protocol results in oligonucleotides ofheretofore unrealized purity, as demonstrated in the Examples below. Thedegree of purity obtainable according to the methods of this aspect ofthe invention is described in detail below.

In addition, we have surprisingly found that synthesis of ultra-pureoligonucleotides according to this aspect of the invention can beconducted without compromising purity or yield using an amount of dimerblock that is much less than has previously been thought possible. Priorart methods, such as those disclosed by Wolter et al., supra, have useda large excess of dimer (often in the range of tens of equivalents) inorder to drive the reaction to completion and ensure high yield. We havesurprisingly found, however, that fewer than about 6 equivalents can beused and still obtain high yields of highly pure oligonucleotide.Accordingly, in a preferred embodiment of this aspect of the invention,≦6 equivalents of dimer are used in each coupling step. More preferably˜2 equivalents of dimer are used per coupling step.

Traditional methods of synthesis, be it using monomers or dimers, employa capping step during each synthetic cycle to block unreacted reactivesites, thereby minimizing subsequent addition of nucleotides to thesesites rather than to the nascent oligonucleotide. Capping was thought tobe a necessary step to achieve high yields of pure oligonucleotides. Wehave surprisingly found, however, that synthesis of oligonucleotidesusing dimer blocks as disclosed herein can be conducted without thecapping step. The elimination of the capping step also results in atremendous savings in time and money without sacrificing yield.

Accordingly, in another preferred embodiment of this aspect of theinvention, dimer block synthesis of oligonucleotides is conductedwithout the capping step.

Purification of oligonucleotides prepared by dimer block synthesis maybe accomplished using two approaches. When the purity of the crudeoligonucleotide is relatively low, the crude oligonucleotide ispreferably purified by RPC followed by IEX. Oligonucleotides having aN:N-1 ration of 100:0 (i.e., non-detectable amounts of N-1oligonucleotide) can be obtained.

Alternatively, for crude products of higher purity, RPC can be omittedand the crude oligonucleotide purified by IEX to give an oligonucleotidewith an N:N-1 ratio of 100:0 (i.e., no detectable N-1 impurity).

As evidenced in Table 5, crude products having a range of purities havebeen obtained. Depending on the purity of the crude product, a two-stepchromatographic purification (RPC followed by IEX) or a single-stepchromatographic purification (IEX only) may be employed. As evidenced inTable 4, crude products of lower purity (e.g., 15 and 300 μmol scale,synthesized with capping) are purified by RPC to yield an intermediatematerial whose purity is approximately equivalent to that of a crudeproduct having high purity as originally synthesized (e.g., 300 μmolscale, synthesized without capping). In the current comparison, relativepurities of crude products are clearly differentiated on the basis ofIEX and CE analyses. Relative purities of feedstocks taken forpurification by IEX are well defined by IEX analysis of thedetritylation mixtures.

As evidenced in Table 4, the single-step chromatographic purificationprovides higher overall recoveries. Taken together, the two approaches(i.e., the one step and two step approaches) provide robust andversatile tools for purification of oligonucleotide prepared by dimersynthesis. Crude products having a range of purities can beaccommodated.

A strongly preferred technique in achieving required purity is rigorousscreening of trial pools by CE analysis. During initial work at 15 μmolscale (infra), this technique was not employed. In that work, productpurity was 97% by CE and (N+x) content was 2.4%. In subsequent work,rigorous use of CE analysis was incorporated and product purities≧98% byCE were achieved, and (N+x) content was reduced to 0.3% or less.

The data indicate that dimer synthesis, performed with or withoutcapping, provides a crude product which can be brought to≧98% finalpurity. Achievement of 98+% product purity can be seen as arising fromthe following factors:

(1) Use of dimer synthesis. This approach has the inherent potential toentirely eliminate (N-1) impurity. Such impurity presents the greatestchallenge to chromatographic purification.

(2) Use of high-resolution preparative chromatography.

(3) Rigorous use of capillary electrophoresis to analyze trial poolsprepared from chromatographic fractions.

The synthetic methods of the invention can be conducted at both small(e.g., 1 μmol) scale as well as large (e.g., ≧100 μmol) scale, resultingin oligonucleotide product with similar purity and yield.

In any of the embodiments according to this aspect of the invention,both monomers of any dimer block used in the synthetic method can bedeoxyribonucleotides, or one can be a deoxyribonucleotide and the othercan be a ribonucleotide.

In a preferred embodiment of the present method, dimer phosphorothioatesare employed to yield an oligonucleotide comprising entirelyphosphorothioate internucleoside linkages. Oligonucleotidephosphorothioates with a PO impurity level of less than 0.5%, preferably0.3%, more preferably ≦0.4%, and most preferably non-detectable by ³¹ PNMR can be obtained.

In a preferred embodiment, the purity of the crude oligonucleotidephosphorothioate (i.e., before purification by chromatographic means)produced by the method according to the invention is ≧75% as determinedby CE. Preferably in this embodiment:

a) the N-1 content is non-detectable;

b) the N-1 content is non-detectable, the N-2 content is <6%, the N-xcontent for x>2 is ≦15% and the PO content is ≦0.6%;

c) the N-1 content is non-detectable and the N-2 content is <1%;

d) the N-1 content is <2%;

e) the N-1 content is less than or equal to 0.5%; or

f) the N+x content is <8%.

Another preferred dimer block is one having a phosphotriesterinternucleotide linkage.

Those skilled in the art will recognize that this approach also allowsthe convenient synthesis of mixed phosphate backbone oligonucleotides,e.g., oligonucleotides having any combination of one or morephosphorothioate, alkyl-/arylphosphonothioate, phosphodiester, and/oralkyl-/arylphosphonate linkages.

The method according to this aspect of the invention provides severaladvantages over monomeric synthesis of oligonucleotides. First, sincehalf as many assembly cycles are required, the total assembly time isreduced by half, which, for large scale synthesis, can be a saving of 12hours or more for a single oligonucleotide. This reduction in time alsoresults in fewer side reactions, since partially assembledoligonucleotides are exposed to chemicals for a shorter time. The methodalso facilitates purification of oligonucleotides by increasing theproportion of full length oligonucleotides, since that proportion variesinversely with the number of cycles performed. Finally, the methodreduces cost of synthesis by cutting solvent consumption by half and byallowing one half of the total synthesis to be carried out usinginexpensive solution phase chemistry. The present method extends theseadvantages to oligonucleotides having exclusively phosphorothioate,alkylphosphonate, phosphoramidate, or alkylphosphonothioate linkages aswell as to oligonucleotides having any combination thereof.

Ultra-Pure Oligonucleotides

The methods of the third aspect of the invention produceoligonucleotides of heretofore unobtainable purity. Accordingly, in afourth aspect, the invention provides oligonucleotides produced by themethods of the third aspect of the invention and having a puritydescribed below. Oligonucleotide phosphorothioates are a preferredoligonucleotide according to this aspect of the invention. As usedherein, the term oligonucleotide phosphorothioate is an oligonucleotidehaving all phosphorothioate internucleotide linkages.

Oligonucleotide product of length N contains two major types ofimpurities, size impurity (type "A") and composition impurity (type "B")such that the "total purity" of a population of oligonucleotides can bedefined by:

    total purity=100% -(% impurity A+% impurity B)

where "% impurity A" is the total percentage of oligonucleotides oflength other than N (i.e., % (N+x)+% (N-x), where x is an integer otherthan 0 and N+x represents all oligonucleotides of length greater than Nnucleotides and N-x represents all oligonucleotides of length less thanN nucleotides). Type A impurities are detectable and quantitatable bycapillary electrophoresis.

"% impurity B" relates to oligonucleotides having all modified (i.e.,non-phosphodiester) linkages and is the total percentage ofoligonucleotides of length N having one or more phosphodiesterinternucleotide linkages in place of the modified linkage. In thepreferred oligonucleotide phosphorothioates, the type B impurity is anoligonucleotide having at least one phosphorothioate linkage replaced bya phosphodiester linkage. Type B impurities (commonly called "PO"impurities) are detectable along with N-x' impurity by ion exchangechromatographic (IEX) analysis of oligonucleotide (DMT-off form). Thepeak in the IEX chromatogram corresponding to the PO impurity plus theN-x' impurity appears just before the peak of the desired N-meroligonucleotide. With reference to this impurity peak, the term N-x'refers to the sum of N-2 plus N-3 and/or N-4. (The terms N-2, N-3, andN-4 as employed in this calculation are defined further below.) Asdefined, the type B (or PO) impurity is estimated for oligonucleotidesby using the following formula:

    %PO=[α.sub.PO/N-x'].sub.IEX [α.sub.N-2 +α.sub.N-3,4)].sub.CE

where ##EQU1## and A_(i) is the area under peak "i" of the chromatogram,the sum in the denominator of the definition of "α" is over all peaks inthe chromatogram, and the subscripts "IEX" and "CE" indicate thechromatographic technique from which the data were obtained. α_(PO/N-x)refers to the peak immediately preceding the peak corresponding to the"N" oligomer in IEX and has contributions from both N-x oligonucleotidesand N oligonucleotides having a PO internucleoside linkage. [α_(N-)2]_(CE) corresponds to oligonucleotides of length N-2, and [α_(N-3),4]_(CE) corresponds to the peak immediately preceding the N-2 peak in CEchromatograms and is believed to arise primarily from oligonucleotidesof length N-3 and/or N-4. The above formula for calculating estimatedvalues for %PO was applied only to product purified by IEX (DMT-off).Results appear in Table 4. A second approach was applied to calculatingestimated values for %PO in crude oligonucleotides (DMT-on): ##EQU2##Wherein [A_(PO/N-x') ]_(IEX) refers to the area of the peak immediatelypreceding that corresponding to the DMT-on form of the Noligonucleotide. This calculation tends to over-estimate the PO contentas it does not subtract the contribution from N-x' impurities. Thus,this approach provides a conservative estimate of purity with respect toPO content. Values based on this calculation appear in Table 5. For bothpure and crude oligonucleotide, %PO can also be determined by ³¹ P.

Other calculations employed herein are based on the PO/N-x' peak.%DMT-on (by IEX) is calculated as follows:

    %DMT-on=[α.sub.N +α.sub.PO/N-x' ].sub.IEX-DMT-n

where α_(PO/N-x') refers to the peak immediately preceding that for theDMT-on form of the N oligonucleotide. The calculation provides a usefulestimate of combined phosphorothioate and phosphodiester forms of DMT-onoligonucleotide, N-oligomer. No correction is made for N-x' content inthe α_(PO/N-x') peak. Results based on this calculation appear in Tables4 and 5.

In a preferred embodiment, oligonucleotide phosphorothioates accordingto this aspect of the invention have a total purity of 98% or more. Morepreferably, the total purity is greater than 99%. Preferably in theseembodiments

a) the N-1 content is non-detectable;

b) the N-1 content is non-detectable, the N-2 content is less than 1%,and the N-x content for x>2 is less than 2%;

c) the N-1 content is non-detectable and the N-2 content is less than1%;

d) the N-1 content is non-detectable and the N-x content for x>1 is lessthan 2%;

c) the N-1 content is <2%

f) the N-1 content is less than or equal to 0.5%;

g) the N+x content is <2%;

h) the N+x content is <1%; or

i) the N+x content is <0.5%.

Unless expressly indicated otherwise, all percentages ofoligonucleotides of a particular length mean percentages as measured bycapillary gel electrophoresis. As used herein, "non-detectable" meannon-detectable by capillary gel electrophoresis, which can detect asingle oligonucleotide size impurity (e.g., N-x and N+x) down to 0.15%.Typically in capillary electrophoresis, the noise level is ˜20-30 mV.The minimum detectable peak has a S/N of ˜3:1, or about 60 mV.

Oligonucleotides according to this aspect of the invention can be ofessentially any conventionally synthesizable length and can be madeaccording to the third aspect of the invention. Preferably,oligonucleotides according to this aspect of the invention are 50 orfewer nucleotides in length, more preferably, 30 or fewer, and mostpreferably of length of from about 15 to about 30 nucleotides.

In yet another embodiment of this aspect of the invention, theoligonucleotide phosphorothioates have less than 0.5% phosphodiestercontent (i.e., the number of phosphodiester linkages comprises less than0.5% of the number of phosphorothioate linkages as measured by ³¹ PNMR). Preferably, the PO content is less than or equal to 0.3%. Morepreferably, the phosphodiester content is less than or equal to0.03%-0.04%, the lower limit of detection of ³¹ P NMR. Even morepreferably, the PO content is non-detectable by ³¹ P NMR.

The following examples are intended to further illustrate certainpreferred embodiments of the methods according to the invention, and arcnot intended to be limiting in nature.

EXAMPLES Example 1

Solution Phase Synthesis Of 5'-O-dimethoxytrityl-thymidine-3'-O-methylphosphorothioate-5'-O-N⁴ -benzoyl-2'-deoxycytidine

The synthesis steps for this protected dimer block for synthesis ofphosphorothioate containing oligonucleotides are shown below: ##STR3##wherein (a) is anhydrous acetonitrile and tetrazole, (b) is Beaucagereagent, (c) is tetrahydrofuran and tetrabutyl ammonium fluoride, andDMT is dimethoxytrityl and TBDMS is tert-butyldimethylsilyl.

A mixture of 5'-O-dimethoxytrityl-thymidine-3'-O-methyl N,N-diisopropylphosphoramidite, 1, (1.4 g, 2 mmol) and N⁴-benzoyl-3'-O(tert-butyldimethylsilyl)-2'-deoxycytidine, 2, (0.88g, 2mmol) was dissolved in anhydrous acetonitrilc (25 ml) and a solution oftetrazole (0.45 M, 10 ml) was added. The reaction mixture was stirred atroom temperature for 15 min. Beaucage reagent (0.6 g in anhydrousacetonitrile 15 ml) was added and the mixture was further stirred for 15min. The reaction mixture was evaporated to remove most of theacetonitrile under reduced pressure. The crude reaction product wasextracted with dichloromethane and washed with brine. The organic layerwas dried over Na₂ SO₄ and evaporated to dryness to obtain 3. Product 3was re-dissolved in tetrahydrofuran (16 ml) and treated with a 1 Msolution of tetrabutylammonium fluoride (3 ml, THF) for 15 min. Thereaction mixture was evaporated to almost dryness and partitionedbetween dichloromethane and water. The organic layer was dried over Na₂SO₄ and evaporated to a small volume. The product was purified by columnchromatography using silica gel (2.5×20 cm). The dimer block product, 4,was eluted with 0-7% methanol in dichloromethane (0.5% pyridine); obtain1.3 g (70% yield); not optimized.

³¹ P NMR=70.06.

Example 2

Solution Phase Synthesis Of 5'-O-dimethoxytrityl-N⁴-benzoyl-2'-deoxycylidine-3'-O-methyl phosphorothioate-5'-O-thymidine

The synthesis steps for this dimer block for synthesis ofphosphorothioate-containing oligonucleotides are shown below: ##STR4##wherein (a) is acetonitrile and tetrazole, (b) is Beaucage reagent, and(c) is pyridine acetic acid and hydrazine hydrate. DMT isdimethoxytrityl and Lev is levulinyl.

A mixture of N4-benzoyl5'-O-dimethoxytrityl-2'-deoxycytidine-3'-O-methyl N,N-diisopropylphosphoramidite 6 (1.6 g, 2 mmol) and 3'-O-levulinyl-thymidine, 7, (0.68g, 2 mmol) was dissolved in anhydrous acetonitrile (25 ml) and asolution of tetrazole (0.45 M, 10 ml) was added. The reaction mixturewas stirred for 15 minutes at room temperature. Beaucage reagent (0.6 gin anhydrous acetonitrile 15 ml) was added and the reaction mixture wasfurther stirred for 15 minutes. The reaction mixture was then evaporatedto remove most of the solvent. The crude reaction product was extractedwith dichloromethane and washed with brine to obtain 8. Dichloromethanewas evaporated, the solid residue was re-dissolved in 20 ml pyridine andmixed with 20 ml of 1 M hydrazine hydrate solution in pyridine/aceticacid (3/2). The reaction mixture was stirred for 5 min. The reactionmixture was then cooled on an ice-bath and 4 ml acetyl acetone was addedto quench the excess amount of hydrazine hydrate. The mixture wasevaporated to a small volume and then directly applied to silica gelcolumn chromatography (2.5×25 cm). The dimer block product, 9, waseluted by using 0-7% methanol in dichloromethane (0.5% pyridine) toobtain 1.25 g (67% yield). ³¹ P NMR=69.86.

Example 3

Solution Phase Synthesis Of 5'-O-dimethoxytrityl-N⁴-benzoyl-2'-deoxycytidine-3'-O-methylphosphorothioate 5'-O-N⁶ -benzoyl-2' deoxyadenosine

The synthetic steps for this dimer block for synthesis ofphosphorothioate-containing oligonucleotides are shown below: ##STR5##wherein (a)-(c) are the same as in Example 2.

A mixture of N⁴-benzoyl-5'-O-dimethoxytrityl-2'-deoxycytidine-3'-O-methylN,N-diisopropyl phosphoramidite, 15, (4 g, 5 mmol) and 3'-O-levulinyl-N⁶-benzoyl-2'-deoxyadenosine, 16, (2 g, 4.4 mmole) was dissolved inanhydrous acetonitrile (18 ml) and a solution of tetrazole (0.45 M, 22ml) was added. The reaction mixture was stirred for 30 minutes at roomtemperature, then treated with Beaucage reagent (1.4 g in anhydrousacetonitrile 25 ml) for 15 minutes. The reaction mixture was thenevaporated to remove most of the solvent. The crude reaction product wasextracted with dichloromethane and washed with brine. Dichloromethanewas evaporated to obtain a solid product, 17, which was then redissolvedin 40 ml pyridine, and 40 ml of 1 M hydrazine hydrate solution inpyridine/acetic acid (3/2) was added. After 7 minutes, the reaction wasquenched with ice and the product was extracted with dichloromethane,then washed with water. The organic layer was dried over sodium sulfateand then co-evaporated with toluene to dryness. The mixture wasre-dissolved in a small volume of dichloromethane and applied to silicagel column chromatography (5×12 cm). The dimer block product, 18, waseluted by using 0-7% methanol in dichoromethane (0.5% pyridine) toobtain 3.8 g (79% yield). ³¹ P NMR=69.84, 69.89.

Example 4

Synthesis ofN-benzoyl-5'-O-dimethoxytrityl-P-cyanoethylthiophosphoryl-2'-deoxycytidylyl-3'-O-[(N,N-diisopropylamino)cyanoethoxyphosphino](3'→5')thymidine (24)

The title phosphoramidite dimer was synthesized as depicted below:##STR6## Synthesis of dimer 22

60 ml of 0.45 M solution of tetrazole in acetonitrile were added to asolution of phosphoramidite 20 (10.0 g, 12.0 mmol) and nucleoside 21(4.0 g, 12.0 mmol) in 150 ml acetonitrile. The reaction was stirred for30 min at room temp. TLC analysis indicated that complete conversion ofthe starting materials to the intermediate phosphite, which was furtheroxidized by adding 88 ml acetonitrile solution of Beaucage Reagent (3.52g, 18.0 mmol). The reaction was stirred for 30 min and then wasevaporated to dryness in vacuo to give dimer 22 as a yellow gum inquantitative yield (13.0 g, ³¹ P NMR, δ 67.0).

Synthesis of dimer 23

Hydrazine monohydrate (2.1 g, 42.0 mmol) was added slowly to an ice-coldsolution of crude dimer 22 (6.63 g, 6.0 mmol) in 65 ml pyridine/aceticacid mixture (3:2). After 20 min, excess hydrazine monohydrate wasquenched with the slow addition of acetyl acetone (11 ml). The reactionwas added to crushed ice and was extracted with methylene chloride (3×50ml). Combined organic layer was dried and evaporated to give a yellowoil, which was chromatographed on flash silica gel. Elution withmethylene chloride/methanol/pyridine (93:5:2) gave dimer 23 as acolorless foam (5.0 g, 82.7% ³¹ P NMR, δ, 67.0).

Synthesis of dimer 24

A solution of dimer 23 (31.5 g, 31.3 mmol) and tetrazole (2.75 g, 39.3mmol) in 150 ml acetonitrile was added to a solution oftetraisopropylphosphorodiamidite (13.0 g, 43.1 mmole) in 60 ml ofacetonitrile. The reaction was stirred at room temperature for 45minutes. It was then cooled in ice and the supernatant solution wasdecanted into another flask. The precipitate was washed with coldacetonitrile three times. The combined acetonitrile solution wasevaporated to dryness. The residue was dissolved in dichloromethane andhexane was added. Supernatant solution was removed and the residual oilwas washed with hexane. The dichloromethane/hexane treatment wasrepeated one more time. The resulting yellow foam was dissolved in ethylacetate containing I% pyridine and was passed quickly through a shortpad of silica gel. Evaporation of the pooled fractions afforded thedimer phosphoramidite 24 as colorless foam (31.7 g, 83.9%, ³¹ P, δ, 67.0(P-V), ˜149.0 (P-III).

Example 5

Determining the purity of phosphorothioate dimers

Authentic samples of CpoT dimer phosphoramidites were made by employingthe same chemistry as that used to synthesize CpsT dimerphosphoramidites, except that t-butyl hydroperoxide was used instead ofBeaucage reagent to oxidize the phosphite intermediate to give thephosphotriester dimer.

Phosphotriester and phosphorothioate dimers can be distinguished by ³¹ PNMR. The phosphoramidite phosphorous signal in both cases appears at ˜δ149. There is a significant difference in the chemical shifts of thephosphorothioate and phosphotriester functions, however.Phosphorothioate triester signal is observed at ˜δ 67.0, whereas thephosphotriester peak appears at ˜δ -2.0. A trace amount of H-phosphonatebyproduct is detected in the phosphorothioate dimer as a doubletcentered at δ 14.5. Thus, ³¹ P NMR can serve as an effective tool indetermining the impurity of phosphotriester in a phosphorothioatetriester compound.

Two samples of phosphorothioate dimer phosphoramidite were spiked with aknown amount of phosphotriester dimer phosphoramidite. A samplecontaining 10% phosphotriester dimer and 90% of phosphorothioate dimerand a second sample constituting 5% phosphotriester dimer and 95%phosphorothioate dimer were prepared and the ³¹ P NMR of the samplesrecorded. ³¹ P NMR spectra of both samples exhibited distinct,well-separated signals at ˜δ 67.0 and ˜δ -2.0, as expected. A 750 timesenlargement of pure phosphorothioate dimer spectrum manifested nodetectable peak at ˜δ 2.0.

Example 6

0.2 μmol scale synthesis of oligonucleotide phosphorothioates

A fluidized bed technique was used for the synthesis of (CT)₁₀ Toligomer on a Perseptive Biosystem Expedite Synthesizer. The design ofthe synthesizer is such that the reagents like activator, amidite, andCap A, Cap B are mixed before entering the column containing resin.There were two capping steps in this method, one before oxidation andanother after oxidation. 37.6 equivalents of dimer were used in eachcoupling step.

In this and all subsequent oligonucleotide syntheses described herein,the dimer phosphoramidite was made as described in Example 4. All otherreagents were purchased from commercial sources and used as received.Fresh solutions of the reagents were made prior to the start of theoligonucleotide synthesis. At the end of the synthesis, resin was driedunder vacuum. The oligonucleotide was cleaved from the support anddeprotected by treating it with ammonium hydroxide at 55° C. overnight.Crude product was analyzed by reverse phase chromatography, ion exchangechromatography and capillary gel electrophoresis.

Detritylation

3% trichloroacetic acid in dichloromethane were used for detritylationin three steps:

a) 0.3 ml was delivered for 8 sec.;

b) 0.3 ml was delivered for 8 sec.; and

c) 0.45 ml was delivered for 30 sec.

Wash

Washing was conducted with acetonitrile. 0.9 ml was delivered for 30sec.

Coupling

The activator used for coupling was 0.45 M 1-H tetrazole inacetonitrile; the amidite was 0.1 M solution of dimer phosphoramidite inacetonitrile. Coupling was conducted as follows:

a) pre-couple wash with acetonitrile--0.075 ml for 1 see;

b) 0.075 ml activator was introduced for 1 sec;

c) 0.075 ml activator was delivered for 1 sec;

d) 0.075 ml amidite was delivered for 1 sec;

e) 0.075 ml activator was introduced for 1 sec;

f) 0.090 ml acetonitrile wash for 2.4 sec;

g) coupling was allowed to continue for 900 sec.

Wash

The resin was washed with 0.27 ml for 900 sec.

Capping

Cap A consisted of 10% acetic anhydride in tetrahydrofuran. Cap B was10% N-methylimidazole, 20% pyridine, and 70% tetrahydrofuran. 0.12 ml ofCap A was delivered for 3 sec followed by delivery of 0.12 ml of Cap Bfor 3 sec.

Wash

The reaction mixture was then washed with 0.375 ml of acetonitrile for17 sec.

Sulfurization

0.45 ml of 2% Beaucage reagent in acetonitrile was delivered for 7 sec.

Wash

The reaction mixture was then washed with 0.3 ml of acetonitriledelivered for 120 sec.

Capping

0.105 ml of each of Cap A and Cap B was delivered for 2 sec.

Wash

The reaction mixture was then washed with 0.9 ml of acetonitriledelivered for 15 sec.

Example 7

15 μmole scale synthesis of oligonucleotide phosphorothioates

The same solutions were used as described previously in this Example tosynthesize the (CT)₁₀ T oligomer on a 15 μmol scale. 5.02 equivalents ofdimer were used in each coupling step.

Detritylation

7.5 ml detritylation solution was delivered for 200 sec.

Wash

Washing was conducted in two steps:

a) 0.75 ml of acetonitrile was delivered for 12.5 sec; and

b) 6.0 ml of acetonitrile was delivered for 100 sec.

Coupling

a) 0.6ml acetonitrile as a pre-couple was added for 16 sec;

b) 0.525 ml activator was delivered for 14 sec;

c) 0.375 ml amidite was delivered for 10 sec;

d) 0.375 activator was delivered for 10 sec;

e) pause for 60 sec;

f) 0.3 ml activator was delivered for 30 sec;

g) 0.6 ml acetonitrile wash for 10 sec;

h) 0.375 ml amidite was delivered for 10 sec;

i) 0.375 ml activator was delivered for 10 sec;

j) pause for 60 sec;

k) 0.3 ml activator was delivered for 30 sec;

l) coupling was allowed to continue for an additional 900 sec.

Wash

Washing was conducted in two steps:

a) 1.5 ml of acetonitrile was delivered for 40 sec; and

b) 1.5 ml of acetonitrile was delivered for 25 sec.

Capping

a) 1.125 ml Cap A was delivered for 30 sec;

b) 1.125 ml Cap B was delivered for 30 sec.

Wash

a) 0.225 ml acetonitrile wash for 40 sec, followed by

b) 1.5 ml acetonitrile wash for 25 sec.

Sulfurization

a) 1.875 ml of Beaucage solution was added over 50 sec and allowed toreact for 60 sec.

Wash

a) 1.5 ml of acetonitrile wash was added for 25 sec.

Capping

a) 0.750 ml Cap A was delivered for 20 sec.

b) 0.750 ml Cap B was delivered for 20 sec.

Wash

a) 5.1 ml acetonitrile was added for 85 sec.

Example 8

300 μmole scale synthesis of oligonucleotide phosphorothioates

Syntheses of (CT)₁₀ T oligomer was conducted on a 300 μmol scale on aPharmacia OligoPilot II Synthesizer. CPG-T was purchased from GlenResearch (Sterling, Va.).

Flow-through type column reactor was used for the synthesizer. CPG-Tsupport was packed in the column. The amount and the rate at which thereagent was delivered to the reactor column depended on the scale of thesynthesis and the size of the column.

In one synthesis, 10.5 g CPG-T (29.0 μmol/g) was used in a 46 ml sizecolumn. Two equivalents of dimer phosphoramidate were used in eachcoupling step for all 300 μmol scale syntheses. The synthesis cycleconsisted of the following steps:

Detritylation

3% dichloroacetic acid in dichloroethane was passed through the solidsupport for 3 minutes at a rate of 75 ml/min.

Wash

Washing was conducted in two steps:

a) Acetonitrile was passed through the column for 6 min at a rate of 75ml/min.

b) Acetonitrile was passed through the column for 1.92 min at a rate of75 ml/min.

Coupling

The activator solution and the amidite solution were injected inalternate fashion. The activator solution was introduced to the reactorfor 1 min at a rate of 36 ml/min. This process was repeated eight times.The amidite solution was introduced for 0.2 min at a rate of 3.8 ml/min.This process was also repeated eight times. Again the activator solutionwas pumped for 0.1 min at a rate of 36 ml/min. The line to the reactorwas washed with acetonitrile for 0.1 min at a rate of 4 ml/min. Thisactivity was repeated 8 times. Combined solution of the activator andthe amidite was then circulated in the reactor loop for 6 min at a rateof 25 ml/min.

Wash

The column was washed with acetonitrile for 2 min at a rate of 25ml/min.

Sulfurization

5% Beaucage reagent in acetonitrile was introduced to the column for 0.6min at a rate of 48 ml/min. The line was washed with acetonitrile for0.1 min at a rate of 15 ml/min. The Beaucage solution was circulated inthe loop for 5 min at a rate of 50 ml/min.

Wash

The column was washed with acetonitrile for 1 min at a rate of 50ml/min.

Capping

The two capping solutions used comprised:

Cap A: 20% N-methylimidazole in acetonitrile; and

Cap B: 20% acetic anhydride, 30% sym-collidine, 50% acetonitrile.

Cap A and Cap B solutions were pumped into the reactor alternatively.Cap A solution was introduced for 0.1 min at a rate of 18 ml/min. Thisaction was repeated eight times. Cap B solution was also injected for0.1 min at a rate of 18 ml/min. This process was repeated eight times.

Wash

a) The first wash step was done for 4.17 min at a rate of 14.4 ml/min.

b) This next wash step was performed for 1.28 min at a rate of 75ml/min.

Example 9

300 μmole scale synthesis of oligonucleotide phosphorothioates withoutcapping This synthesis of the (CT)₁₀ T oligomer was conducted using 8.0g of CPG-T (38.0 μmol/g) in a 24 ml size reactor. The reagents used forthis synthesis were the same as those described in Example 8, but, asseen in the protocol below, a capping step was not employed. Twoequivalents of dimer were used for each coupling step. The synthesiscycle is described below.

Detritylation

The detritylation solution was passed through the solid support for 3min a rate of 50 ml/min.

Wash

a) The column was washed with acetonitrile. Acetonitrile was passedthrough the column for 6 min at a rate of 50 ml/min.

b) This wash utilized acetonitrile for 1.44 min at a rate of 50 ml/min.

Coupling

The activator solution and the amidite solution were injected inalternate fashion. The activator solution was introduced to the reactorfor 0.1 min at a rate of 24 ml/min. This process was repeated six times.The amidite solution was introduced for 0.2 min at a rate of 4.8 ml/min.This process was also repeated six times. Again the activator solutionwas pumped for 0.1 min at a rate of 24 ml/min. The line to the reactorwas washed with acetonitrile for 0.1 min at a rate of 4 ml/min. Thisactivity was repeated eight times. Combined solution of the activatorand the amidite was then circulated in the reactor loop for 6 min at arate of 20 ml/min.

Wash

The column was washed with acetonitrile for 1 min at a rate of 20ml/min.

Sulfurization

Beaucage solution was introduced to the column for 0.6 min at a rate of24 ml/min. The line was washed with acetonitrile for 0.2 min at a rateof 15 ml/min. The Beaucage solution was circulated in the loop for 4.6min at a rate of 28.8 ml/min.

Wash

The column was washed with acetonitrile for 1 min at a rate of 24ml/min.

Capping

The program for capping was not changed. The Cap A and Cap B bottleswere filled with acetonitrile instead of the respective reagents. Thus,the capping step for this protocol becomes the wash step.

Wash

a) This wash step was done for 1.25 min at a rate of 24 ml/min.

b) This step was performed for 0.95 min at a rate of 50 ml/min.

The results of three different syntheses conducted without capping andbefore chromatographic purification are presented as experiment numbers5-7 in Table 5, infra.

Example 10

Purification of (CT)₁₀ T prepared by dimer block synthesis

Oligonucleotides prepared according to the foregoing examples weresubjected to chromatography to purify them further. Products taken forpurification are described in Table 5, lines 3, 4, and 6. These productscorrespond to the three entries for Crude Product in Table 4. As shown,products corresponding to the first two entries in Table 4 were purifiedby RPC followed by IEX. Product corresponding to the third entry inTable 4 was purified using IEX as the sole chromatographic step.Following chromatography, desalted product was prepared byultrafiltration/diafiltration and lyophilization, as required. Productof >98% purity was obtained, as determined by both analytical IEX-HPLCand capillary electrophoresis (CE). ³¹ P NMR was employed to determinePO content.

The purification procedures described below were conducted using crudeproduct prepared at the 15 μmol and 300 μmol scales. Experience obtainedduring work with the 15 μmol scale was applied to the subsequentpurification at the 300 μmol scale resulting in enhanced purity.

Reversed Phase Chromatography

As noted, RPC was used for preliminary purification of crudeoligonucleotide having lower purity, as synthesized. RPC was employed inpurification of crude products described in the first two entries ofTable 4, but was omitted in purifying crude product described in thethird entry of this table. Crude product produced at 15μmole scale (withcapping) is of higher purity than that produced at 300 μmol scale (withcapping). The higher purity of the product obtained at 15 μmol scaleprobably arises from the greater excess of amidite (7-8 fold) employedduring synthesis compared with that employed at 300 μmol scale (2-foldexcess).

RPC was performed using Amberchrom CG-300sd (TosoHaas), a porousstyrenic resin. A combination of gradient and isocratic elution wasemployed. Buffer A was 0.10 M aqueous ammonium acetate. Buffer B was80/20 v/v/acetonitrile/Buffer A.

Chromatography at the 15 μmol scale was performed using a 1.0 cm ID×14.1cm column. Load and wash steps were conducted at 4.0 ml/min; isocraticand gradient elution were conducted at 2.0 ml/min. At the 300 μmolscale, a 2.5 cm ID×20.3 cm column was employed. Two runs were made.During the first run, load and wash steps were performed at 24.5 ml/min;isocratic and gradient elution were conducted at 12.3 ml/min. During thesecond run, the flow rate was 12.3 ml/min throughout.

Feedstock was prepared by addition of Picopure water to the crudeproduct in ammonium hydroxide solution; ammonium acetate was added toprovide a concentration of 0.2 M. At the 15 μmol scale, feedstock had aconcentration of approximately 20 A₂₆₀ units/ml solution. The loadingfactor was approximately 175 A₂₆₀ units/ml bed. At the 300 μmol scale,corresponding values were 53 A₂₆₀ unit/ml solution and 147 A₂₆₀ units/mlbed.

Purification was accomplished using the non-optimized sequence of stepsdisplayed in Table 2. Capping was employed the synthesis of each of thesamples shown.

                  TABLE 2                                                         ______________________________________                                        #   15 μmol scale                                                                           300 μmol scale (run 1)                                                                   300 μmol scale (run 2)                      ______________________________________                                        1   Load         Load          Load                                           2   Wash, 100% (A).sup.1,                                                                      Wash, 100% (A),                                                                             Wash, 100% (A),                                    5.4 CV.sup.3 1.1 CV        1.2 CV                                         3   Grad:.sup.3 0-21%                                                                          Grad: 0-19% (B) @                                                                           Grad: 0-19% (B) @                                  (B).sup.2 @ 1%/min,                                                                        1%/min, 1.1 CV                                                                              1%/min, 1.1 CV                                     1.1 CV                                                                    4   Isocrat.sup.3 21% (B),                                                                     Isocrat 19% (B),                                                                            Isocrat 19% (B),                                   3.4 CV       1.8 CV        1.8 CV                                         5   Grad: 21-72% Grad: 19-32% (B) @                                                                          Grad: 19-31% (B) @                                 (B) @ 1%/min,                                                                              1%/min, 1.5 CV                                                                              1%/min, 1.5 CV                                     9.2 CV                                                                    6   Wash, 100% (B),                                                                            Isocrat 32% (B)                                                                             Isocrat 31% (B) @                                  7.9 CV       1.1 CV        1%/min, 1.2 CV                                 7   --           Grad: 32-55% (B) @                                                                          Grad: 31-55% (B) @                                              1%/min, 2.5 CV                                                                              1%/min, 2.5 CV                                 8   --           Wash, 100% (B),                                                                             Wash, 100% (B),                                                 Approx. 4 CV.sup.4                                                                          Approx. 4 CV.sup.4                             ______________________________________                                         .sup.1 "A" refers to Buffer A (0.1 M ammonium acetate)                        .sup.2 "B" refers to Buffer B (80/20 v/v acetonitrile/Buffer A)               .sup.3 "CV" is column volume, "Grad" is gradient elution, and "isocrat" i     isocratic elution.                                                            .sup.4 The initial 1.5 CV was collected as a chromatographic fraction, th     remainder was diverted to waste.                                         

Pooled chromatographic fractions were drawn from the fourth and fifthsteps in the 15 μmol scale purification and from the sixth step in the300 μmol scale purifications.

Detritylation

Crude product corresponding to the third entry in Table 4 was subjectedto IEX purification without preliminary RPC purification. First,however, crude product in ammonium hydroxide solution was processed on arotary evaporator to remove ammonia prior to detritylation. To theremaining solution, a quantity of pure water was added, as required, toyield a solution having A₂₆₀ ≦50 OD/ml. Finally, a quantity of glacialacetic acid was added to provide a final concentration of 20% V/Vglacial acetic acid. The resulting solution was stirred at roomtemperature for 2.5 hr.

Post-RPC pools were detritylated in a corresponding procedure, butexcluding processing on a rotary evaporator. After addition of glacialacetic acid, the reaction period was 2.25 hr at the 15 μmol scale and2.75 hr at the 300 μmol scale.

After these procedures, the oligonucleotide products were subject toIEX.

Ion Exchange chromatography

IEX was performed using TSK-GEL DEAE-5PW (TosoHaas), a DEAE-substitutedmethacrylic polymer. A combination of gradient and isocratic elution wasemployed. Buffer A was 25 mM TrisCl, pH 7.2. Buffer B was 25 mM TrisClcontaining 2.0 M sodium chloride, pH 7.2.

The detritylated oligonucleotides previously subjected to RPC werepurified as follows:

At the 15 μmol scale, a column 0.66 cm ID×13. 3 cm was employed. Steps1, 2, and 7 (Table 3), were conducted at 1.29 ml/min, while steps 3-6were conducted at 0.86 ml/min. The loading factor was 212 A₂₆₀ units/mibed.

At the 300 μmol scale, a column 2.2 cm ID×19.0 cm was employed. Steps 1,2, and 7 (Table 3) were conducted at 14.3 ml/min, while steps 3-6 wereconducted at 9.5 ml/min. The loading factor was 217 A₂₆₀ units/ml bed.

Crude detritylated oligonucleotide that had not been subjected to RPCwas purified using a column 1.1 cm ID×8.3 cm. Steps 1 and 2 wereconducted at 3.6 ml/min., while steps 3-10 were conducted at 2.4 ml/min(Table 3). The loading factor was 249 A₂₆₀ units/ml bed.

The non-optimized elution steps listed in Table 3 were employed:

                  TABLE 3                                                         ______________________________________                                        15 μmol scale.sup.3                                                                     300 μmol scale.sup.3                                                                     300 μmol scale.sup.4                            (capping)    (capping)     (no capping)                                       ______________________________________                                         1  LOAD         LOAD          LOAD                                            2  Wash, 100% (A).sup.1,                                                                      Wash, 100% (A),                                                                             Wash, 100% (A),                                    7.7 CV.sup.5 6.6 CV        8.5 CV                                          3  Grad..sup.5, 10-40%                                                                        Grad., 20-30% (B) @                                                                         Isocrat..sup.5, 22% (B),                           (B).sup.2 @ 0.5%/min.,                                                                     0.5%/min., 4.0 CV                                                                           3.2 CV                                             11.2 CV                                                                    4  Isocrat. 40% (B),                                                                          Isocrat. 35% (B),                                                                           Isocrat. 30% (B),                                  3.7 CV       3.6 CV        10.5 CV                                         5  Grad., 40-60%                                                                              Grad., 35-52% (B) @                                                                         Isocrat., 36% (B),                                 (B) @ 0.5%/min.,                                                                           0.5%/min., 4.5 CV                                                                           5.1 CV                                             7.4 CV                                                                     6  Isocrat., 75% (B),                                                                         Grad., 52-75% (B) @                                                                         Isocrat. 46% (B),                                  2.2 CV       1%/min., 3.0 CV                                                                             5.9 CV                                          7  Isocrat., 100% (B),                                                                        Isocrat., 75% (B),                                                                          Isocrat., 52% (B),                                 5.0 CV       6.1 CV        7.5 CV                                          8  --           Isocrat., 100% (B),                                                                         Isocrat., 58% (B),                                              4.0 CV        6.6 CV                                          9  --           --            Isocrat., 75% (B),                                                            3.2 CV                                         10  --           --            Isocrat., 100% (B),                                                           5.1 CV                                         ______________________________________                                         .sup.1 "A" refers to Buffer A (0.3 NaOH)                                      .sup.2 "B" refers to Buffer B (0.3 NaOH + 2.0 NaCl)                           .sup.3 detritylated, postRPC pool                                             .sup.4 detritylated crude product                                             .sup.5 CV = column volume, "Grad" is gradient elution, and "Isocrat" is       isocratic elution                                                        

Pooled fractions were drawn from steps 4-7 in the 15 μmol scalesynthesis, steps 6-7 (pool 1) and 5-7 (pool 2) in the 300 μmol scalesynthesis with capping, and steps 5-8 (pool 1) and 6-9 (pool 2) in the300 μmol scale synthesis without capping.

In order to achieve purities≧98% as measured by both IEX-HPLC and CE, itwas necessary to make rigorous use of CE analysis during poolingdecisions. Trial pools were prepared from chromatographic fractions soas to provide 99% and/or 98% purity as determined by IEX-HPLC. Varioussubsets of these trial pools were then prepared using a more restrictedrange of chromatographic fractions. These subsets were then analyzed byCE to assure that they met 98% or 99% purity goals, as required. Thisapproach was successfully employed during purification of crude productsprepared at 300μmole scale. Pools of 98% and 99% purity were isolated,as determined by both IEX-HPLC and CE, and (n+x) values were reduced to0.3% or less. This rigorous use of CE analysis was not employed duringpurification of product prepared at 15 μmole scale. Here, CE purity ofthe product was 97%, and (n+x) content was 2.4%.

Desalting and Lyophilization

As required, desalting was performed by diafiltration using an Amiconstirred cell fitted with a 1000 MWCO membrane. Conductivity of the finaldiafiltrate was reduced to <60 μmho. Lyophilization of the desaltedsolution followed.

The results presented in Table 4 and 5 below are based, in part, ondefinitions and formulae set forth in the "Detailed Description of theInvention" section, particularly the section "UltrapureOligonucleotides." Selected definitions and formulae are presented orrestated in footnotes to these tables for clarity. Additionaldefinitions, formulae, and calculation procedures are presented in thetext immediately following Table 4.

                                      TABLE 4                                     __________________________________________________________________________                                           % Recovery                             Purity                                 After                                                     % Purity by CE      Chromatog.                             Scale                                                                             % DMT-                                                                             % DMT-                                                                             % Purity      Σ N - x                                                                         %  Step (by                               (μmol)                                                                         on RP.sup.11                                                                       on IEX.sup.3                                                                       IEX.sup.4                                                                          N  N - 1                                                                            N - 2                                                                            (X > 2)                                                                           Σ N + x                                                                     PO.sup.12                                                                        IEX)                                   __________________________________________________________________________    Crude Product                                                                 15.sup.1                                                                          82   80   65.sup.9                                                                           74 ND 4.6                                                                              18.8                                                                              3.0 --                                        300.sup.1                                                                         83   75   65.sup.9                                                                           65 0.8                                                                              2.4                                                                              25.3                                                                              7.0 --                                        300.sup.2                                                                         83   88   81.sup.9                                                                           80 0.7                                                                              3.9                                                                              12.0                                                                              3.4 --                                        Purification by RPC (on Amberchrom CG-300sd)                                  15.sup.1                                                                          --   93   73.sup.9                                                                           -- -- -- --  --  -- 70.sup.7                               300.sup.1 :                                                                   Run 1                                                                             --   see pool                                                                           see pool                                                                           -- -- -- --  --  -- 59.sup.7                               Run 2                                                                             --   see pool                                                                           see pool                                                                           -- -- -- --  --  -- 63.sup.7                               Pool                                                                              --   86   76.sup.9                                                                           -- -- -- --  --  -- 61.sup.7                               Runs                                                                          1 + 2                                                                         300.sup.2                                                                         N/A.sup.5                                                                          N/A  N/A  N/A                                                                              N/A                                                                              N/A                                                                              N/A N/A N/A                                                                              N/A                                    Detritylation (Analysis of reaction mixture)                                  15.sup.1                                                                          --   --   71.sup.10                                                                          -- -- -- --  --  --                                        300.sup.1                                                                         --   --   77.sup.10                                                                          87 -- -- --  --  --                                        300.sup.2                                                                         --   --   73.sup.10                                                                          -- -- -- --  --  --                                        Purification by IEX (on DEAE-5PW)                                             15.sup.1                                                                          --   --   98.sup.10                                                                          97 ND.sup.6                                                                         0.8                                                                              ND  2.4 1.0                                                                              88.sup.8                               300.sup.1 :                                                                   Pool 1                                                                            --   --   98.sup.10                                                                          98 ND 0.5                                                                              1.5 0.1 0.2                                                                              76.sup.8                               Pool 2                                                                            --   --   99.sup.10                                                                          99 ND 0.4                                                                              0.5 0.2 0.4                                                                              54.sup.8                               300.sup.2 :                                                                   Pool 1                                                                            --   --   98.sup.10                                                                          99 ND 1.2                                                                              0.2 ND  0.6                                                                              69.sup.8                               Pool 2                                                                            --   --   99.sup.10                                                                          99 ND 0.7                                                                              0.2 0.3 0.0                                                                              47.sup.8                               __________________________________________________________________________     .sup.1 with capping                                                           .sup.2 without capping                                                        .sup.3 [α.sub.N + α.sub.PO/N-x' ].sub.IEX,DMTon                   .sup.4 [α.sub.N ].sub.IEX                                               .sup.5 N/A = not applicable                                                   .sup.6 ND = not detected                                                      .sup.7 % recovery of DMTon form. Calculated as described in text below.       .sup.8 % recovery of product expressed as IEX units. Calculated as            described in text below.                                                      .sup.9 Analysis of DMTon form                                                 .sup.10 Analysis of DMToff form                                               .sup.11 [α.sub.N ].sub.RP                                               .sup.12 Estimated % PO                                                   

Chromatographic recoveries presented in Table 5 are expressed in twoforms. Recovery after RPC purification is expressed with respect to theDMT-on form as percent RPC units recovered. Recovery after IEXpurification is expressed as percent IEX units of product (DMT-off)recovered. RPC units and IEX units present in a quantity of the stock(i.e., load) or in individual or pooled fractions may be calculated asfollows:

(1) calculate the number of A₂₆₀ units in a given volume of solution:

(A₂₆₀ units)=(ml of solution) (A₂₆₀ determined using 1 cm path cuvette)

(2) determine the percent purity by analytical IEX or analytical RPC:

(3) calculate RPC units in a given volume of solution:

    (RPC units)=[(A 260 units) (% DMT-on purities by RPC)]/100

(4) calculate IEX units in a given volume of solution:

    (IEX units)=[(A.sub.260 units)(% purity by IEX)]/100

Using the above values, chromatographic recoveries are calculated:

(1) calculate percent recovery of DMT-on form: ##EQU3## (2) calculatepercent recovery of product as IEX units: ##EQU4##

The overall chromatographic yield for the two step purification (definedas "(% recovery DMT-on by RPC)×(% recovery by IEX)) was 62% at the 15μmol scale and 46% and 29% for pools 1 and 2, respectively, at the 300μmol scale (with capping). The overall chromatographic yield for thesingle-step purification is identical to % recovery by IEX and was thus69% and 47% for pools 1 and 2, respectively, obtained after purificationat the 300 μmol scale (no capping).

Values for estimated %PO in Table 4 were calculated as set forth in the"Detailed Description of the Invention" section, under the heading"Ultrapure Oligonucleotides." Purified product described in the finalentry of Table 4 (Pool2, 300 μmol scale, without capping) was alsoanalyzed by ³¹ P NMR to determine PO content. In this analysis no PO wasdetected.

Additional 21-mer oligonucleotides were synthesized according to theforegoing protocols. The results are presented in Table 5. Theoligonucleotide was obtained in the range of 65-81% as detected bycapillary electrophoresis (see Table 5). Interestingly, in experiment 2,when 75% of the 21-mer was observed, the N-1 content was undetectablysmall. The average stepwise coupling yield in experiment 2 was 97.2%.Ion-exchange chromatography estimates of the phosphodiester content perlinkage in the phosphorothioate 21-mer was generally below 1.0%.

The average stepwise coupling yield for the 15.0 μmol scale synthesiswas 97.0%, whereas for the 300 μmol scale synthesis it was 97.2%.

                                      TABLE 5                                     __________________________________________________________________________    Scale          % IEX                                                                              % CE                                                      #  (μmol)                                                                         ODs % RP                                                                              DMT-on                                                                             N  N - 1                                                                            N - 2                                                                             N + x                                                                            % PO (IEX)                                   __________________________________________________________________________     1*                                                                              0.2   24                                                                              84  86   65 10.6                                                                              3.11                                                                             5.7                                                                              0.7                                          2  0.2   23                                                                              87  81   75 ND 5.4 4.9                                                                              1.2                                          3  15.0                                                                               1700                                                                             82  80   74 ND 4.6 3.0                                                                              0.9                                          4  300 30000                                                                             83  75   65 0.8                                                                              2.4 7.0                                                                              0.7                                          5  289 30894                                                                             79  72   75 1.1                                                                              2.3 7.4                                                                              0.4                                          6  304 33360                                                                             83  87   80 0.7                                                                              3.9 3.4                                                                              0.4                                          7  304 33583                                                                             87  87   81 0.6                                                                              3.1 3.9                                                                              0.4                                          __________________________________________________________________________     *monomer synthesis                                                            ND = not detectable                                                      

We claim:
 1. A method of manufacturing a N-mer oligonucleotidecomposition of ultrahigh purity, the method comprisinga) synthesizingthe oligonucleotide with dimer blocks using phosphoramidite chemistry,said synthesis comprising contacting a nascent oligonucleotide with 6 orfewer equivalents of a phosphoramidite dimer block, oxidation,detritylation, and repeating each the foregoing steps in this part (a)until the N-mer oligonucleotide is obtained; b) subjecting thefull-length, N-mer oligonucleotide synthesized in part (a) to (i)ion-exchange chromatography or to (ii) either reversed phase orhydrophobic interaction chromatography followed by ion-exchangechromatography; c) pooling chromatographic fractions from part (b)having equal to or greater than 99% total purity of N-meroligonucleotide.